本文選題：強(qiáng)直性脊柱炎(AS) + 調(diào)節(jié)性T細(xì)胞(Treg　； 參考：《第四軍醫(yī)大學(xué)》2017年博士論文

【摘要】：目的強(qiáng)直性脊柱炎(ankylosing spondylitis,AS)是一種主要累及脊柱、骶髂關(guān)節(jié)及外周關(guān)節(jié)的慢性免疫性炎性疾病,其根本發(fā)病機(jī)制尚不清楚。近年來(lái)的研究表明,CD4+T細(xì)胞亞群調(diào)節(jié)性T細(xì)胞(regulatory T cells,Treg cells)、輔助性T細(xì)胞17(T-helper cells 17,Th17 cells)以及輔助性T細(xì)胞1(T-helper cells 1,Th1 cells)等均可能參與AS發(fā)病。其中CD4+CD25+FoxP3+Treg細(xì)胞是體內(nèi)重要的抑制性T細(xì)胞,它們可以在體內(nèi)和體外抑制多種免疫細(xì)胞功能,從而維持自身免疫耐受及免疫平衡。如果實(shí)驗(yàn)動(dòng)物Treg細(xì)胞發(fā)育異常,會(huì)發(fā)生嚴(yán)重的自身免疫性疾病。既往文獻(xiàn)表明Treg細(xì)胞參與多種自身免疫性疾病的發(fā)生,如多發(fā)性硬化(multiplesclerosis,MS)與1型糖尿病(type1 diabetes,T1D)等。目前各CD4+T細(xì)胞亞群,尤其是Treg細(xì)胞與AS發(fā)生發(fā)展的關(guān)系尚未明確。為了研究Treg細(xì)胞等CD4+T細(xì)胞亞群參與AS發(fā)病的分子機(jī)制,我們進(jìn)行了本研究。方法收集活動(dòng)性及穩(wěn)定性AS患者外周血,分離外周血單個(gè)核細(xì)胞(peripheral blood mononuclear cells,PBMCs),應(yīng)用流式細(xì)胞術(shù)從PBMCs中分選SYTOX Green-CD4+CD25-CD45RA+幼稚T細(xì)胞(na?ve T cells,Tn cells)和SYTOX Green-CD4+CD25high Treg細(xì)胞。第一部分檢測(cè)Tn細(xì)胞的增殖、凋亡及向CD4+T細(xì)胞亞群Th17細(xì)胞誘導(dǎo)分化的能力。取分選的Tn細(xì)胞,用5(6)-羧基二乙酸熒光素琥珀酰亞胺酯(5,6-carboxyfluorescein succinimidyl ester,CFSE)標(biāo)記后,加入抗-CD3/CD28磁珠培養(yǎng),于細(xì)胞培養(yǎng)第5天收集細(xì)胞,用流式細(xì)胞儀檢測(cè)并分析Tn細(xì)胞增殖;取分選的Tn細(xì)胞,加入抗-CD3/CD28磁珠培養(yǎng),于細(xì)胞培養(yǎng)24小時(shí)和72小時(shí)分別收集細(xì)胞,用AnnexinⅤ和PI將細(xì)胞染色后,15分鐘內(nèi)用流式細(xì)胞儀檢測(cè)并分析早期與晚期凋亡的Tn細(xì)胞比例;取PBMCs,用流式細(xì)胞儀檢測(cè)IL-23R表達(dá)情況,同時(shí)取分選的Tn細(xì)胞,加入抗-CD3/CD28磁珠與Th17細(xì)胞誘導(dǎo)分化細(xì)胞因子重組人白細(xì)胞介素6(recombinant human interleukin 6,rhIL-6),重組人轉(zhuǎn)化生長(zhǎng)因子β1(recombinant human transforming growth factorβ1,rhTGF-β1),重組人白細(xì)胞介素1β(recombinant human interleukin 1β,rhIL-1β),重組人白細(xì)胞介素23(recombinant human interleukin23,rhIL-23)培養(yǎng),于細(xì)胞培養(yǎng)第三天和第七天檢測(cè)Th17細(xì)胞誘導(dǎo)分化比例。第二部分應(yīng)用細(xì)胞流式染色法分析PBMCs中Treg細(xì)胞等CD4+T細(xì)胞亞群比例及其相應(yīng)的平均熒光強(qiáng)度(mean fluorescence intensity,MFI)。取PBMCs,加入適量相應(yīng)的細(xì)胞外、細(xì)胞內(nèi)以及細(xì)胞核內(nèi)流式抗體染色后,用流式細(xì)胞儀檢測(cè)并分析Treg細(xì)胞,Th17/Th1細(xì)胞比例及相應(yīng)MFI。第三部分檢測(cè)Treg細(xì)胞對(duì)Tn細(xì)胞增殖的抑制功能。取分選的Tn細(xì)胞,用CFSE標(biāo)記后單獨(dú)或者與分選的Treg細(xì)胞共培養(yǎng),同時(shí)加入抗-CD3/CD28磁珠,于細(xì)胞培養(yǎng)第5天收集細(xì)胞,用流式細(xì)胞儀檢測(cè)并分析Treg細(xì)胞對(duì)Tn細(xì)胞增殖的抑制功能;同時(shí)用酶聯(lián)免疫分析法(enzyme-linked immunosorbent assay,ELISA)法檢測(cè)Treg細(xì)胞與Tn細(xì)胞共培養(yǎng)上清液中轉(zhuǎn)化生子因子β(transforming growth factorβ,TGF-β)、顆粒酶B(Granzyme B)、白細(xì)胞介素10(interleukin 10,IL-10)和白細(xì)胞介素2(interleukin 2,IL-2)等細(xì)胞因子的濃度。第四部分檢測(cè)Treg細(xì)胞IL-2信號(hào)通路(包括IL-2使用及其下游信號(hào)通路)是否異常。取分選的Tn細(xì)胞單獨(dú)或者與Treg細(xì)胞共培養(yǎng),同時(shí)加入抗-CD3/CD28磁珠,細(xì)胞培養(yǎng)第5天收集細(xì)胞,提取mRNA并反轉(zhuǎn)錄為cDNA,應(yīng)用實(shí)時(shí)定量PCR(Real-Time quantitative polymerase chain reaction,qPCR)檢測(cè)Tn細(xì)胞IL-2 mRNA表達(dá)及Treg細(xì)胞相關(guān)細(xì)胞因子m RNA表達(dá)。同時(shí)取分選的Treg細(xì)胞,在IL-2刺激后不同時(shí)間點(diǎn)檢測(cè)其信號(hào)轉(zhuǎn)導(dǎo)與轉(zhuǎn)錄激活因子5(signal transducer and activator of transcription 5,STAT5)的磷酸化水平。第五部分檢測(cè)Treg細(xì)胞FoxP3基因中保守的非編碼序列(the conserved non-coding DNA sequence,CNS)2表觀遺傳學(xué)調(diào)控是否異常。取分選的Treg細(xì)胞,提取基因組DNA,應(yīng)用亞硫酸氫鈉測(cè)序分析其FoxP3基因CNS2區(qū)域10個(gè)Cp G島甲基化水平。數(shù)據(jù)采用GraphPad Prism 7.6.1軟件進(jìn)行統(tǒng)計(jì)描述和分析。p0.05為差異具有統(tǒng)計(jì)學(xué)意義。結(jié)果1.與健康對(duì)照者相比,活動(dòng)性AS患者Tn細(xì)胞增殖、凋亡及向CD4+T細(xì)胞亞群Th17細(xì)胞誘導(dǎo)分化功能均沒(méi)有明顯改變。2.與健康對(duì)照者相比,穩(wěn)定性AS患者與活動(dòng)性AS患者PBMCs中Treg細(xì)胞,Th17/Th1細(xì)胞等CD4+T細(xì)胞亞群比例均沒(méi)有明顯改變。但活動(dòng)性AS患者Treg細(xì)胞FoxP3 MFI顯著下降,提示Treg細(xì)胞功能異常。3.與健康對(duì)照者相比,活動(dòng)性AS患者Treg細(xì)胞不能有效抑制Tn細(xì)胞增殖,說(shuō)明活動(dòng)性AS患者Treg細(xì)胞功能有缺陷。4.與健康對(duì)照者相比,活動(dòng)性AS患者Treg細(xì)胞與Tn細(xì)胞共培養(yǎng)上清液中IL-2濃度增高,同時(shí)qPCR檢測(cè)結(jié)果表明AS患者Tn細(xì)胞并沒(méi)有比健康對(duì)照者Tn細(xì)胞分泌更多IL-2,在共培養(yǎng)體系中的Tn細(xì)胞利用IL-2也并未減少,這些結(jié)果表明活動(dòng)性AS患者Treg細(xì)胞可能存在IL-2利用缺陷,從而導(dǎo)致其使用IL-2減少。進(jìn)一步檢測(cè)IL-2刺激后不同時(shí)間點(diǎn)Treg細(xì)胞STAT5磷酸化水平,發(fā)現(xiàn)活動(dòng)性AS患者在IL-2刺激后Treg細(xì)胞STAT5表達(dá)增加,但是其磷酸化水平卻沒(méi)有相應(yīng)增加,這說(shuō)明Treg細(xì)胞存在STAT5磷酸化不足或者不穩(wěn)定。以上結(jié)果表明活動(dòng)性AS患者Treg細(xì)胞IL-2信號(hào)通路確實(shí)存在異常,這可能會(huì)影響Treg細(xì)胞功能。5.活動(dòng)性AS患者Treg細(xì)胞FoxP3基因表觀遺傳學(xué)調(diào)控異常,其CNS2區(qū)域Cp G島甲基化水平升高。結(jié)論本課題首先研究了Treg等CD4+T細(xì)胞亞群是否參與AS發(fā)病。在這部分中我們檢測(cè)了活動(dòng)性AS患者CD4+T細(xì)胞亞群分化前Tn細(xì)胞的增殖,凋亡以及向CD4+T細(xì)胞亞群Th17細(xì)胞誘導(dǎo)分化的能力,并沒(méi)有發(fā)現(xiàn)異常。我們進(jìn)一步分析了AS患者PBMCs中CD4+T細(xì)胞亞群Treg細(xì)胞以及Th17/Th1細(xì)胞的比例及相應(yīng)MFI,首次發(fā)現(xiàn)活動(dòng)性AS患者Treg細(xì)胞FoxP3 MFI明顯下降,提示Treg細(xì)胞功能異常。因此我們進(jìn)一步檢測(cè)了Treg細(xì)胞對(duì)Tn細(xì)胞的增殖抑制功能,結(jié)果發(fā)現(xiàn)活動(dòng)性AS患者Treg細(xì)胞不能有效抑制Tn細(xì)胞增殖。這些結(jié)果證明活動(dòng)性AS患者Treg細(xì)胞確實(shí)存在功能缺陷。在發(fā)現(xiàn)活動(dòng)性AS患者Treg細(xì)胞存在功能缺陷的基礎(chǔ)上,本課題進(jìn)一步重點(diǎn)研究了Treg細(xì)胞功能缺陷的可能機(jī)制。一方面我們檢測(cè)了活動(dòng)性AS患者Treg細(xì)胞IL-2利用及IL-2下游信號(hào)通路是否異常,結(jié)果發(fā)現(xiàn)Treg細(xì)胞利用IL-2能力下降,在IL-2充足時(shí)也不能很好的利用環(huán)境中的IL-2,同時(shí)IL-2下游重要信號(hào)分子STAT5磷酸化水平相對(duì)不足或者不穩(wěn)定。另一方面我們檢測(cè)了活動(dòng)性AS患者Treg細(xì)胞FoxP3基因表觀遺傳學(xué)調(diào)控,發(fā)現(xiàn)其CNS2區(qū)域CpG島甲基化水平異常升高。IL-2信號(hào)通路異常與CpG島甲基化水平升高共同引起Treg細(xì)胞抑制功能異常,最終導(dǎo)致AS發(fā)生。目前,靶向Treg細(xì)胞治療自身免疫性疾病和炎性疾病方興未艾,已取得突破性進(jìn)展,目前應(yīng)用不同方法恢復(fù)Treg細(xì)胞數(shù)量以及功能而治療系統(tǒng)性紅斑狼瘡(systemic lupus erythematosus,SLE)、MS、銀屑病和潰瘍性結(jié)腸炎(ulcerative colitis,UC)等疾病均有報(bào)道。本研究的發(fā)現(xiàn)為臨床上靶向恢復(fù)Treg細(xì)胞功能治療AS提供了理論依據(jù),也為從根本機(jī)制上治療AS提供了更多方向與選擇。
[Abstract]:Objective ankylosing spondylitis (AS) is a chronic inflammatory inflammatory disease involving the spinal, sacroiliac and peripheral joints. Its underlying pathogenesis is still unclear. Recent studies have shown that the CD4+T cell subgroup (regulatory T cells, Treg cells), auxiliary T cell 17 (T-helper 17) H17 cells) and auxiliary T cells 1 (T-helper cells 1, Th1 cells) may all participate in the pathogenesis of AS, and CD4+CD25+FoxP3+Treg cells are important inhibitory T cells in the body. They can inhibit a variety of immune cell functions in vivo and in vitro so as to maintain their immune tolerance and immune balance. If experimental animals Treg cell development Often, there will be a serious autoimmune disease. Previous literature shows that Treg cells are involved in a variety of autoimmune diseases, such as multiple sclerosis (multiplesclerosis, MS) and type 1 diabetes (type1 diabetes, T1D). At present, the relationship between the CD4+T cell subsets, especially the Treg cells and the development of AS is not clear. In order to study Treg fine The molecular mechanism of cell like CD4+T cell subsets involved in the pathogenesis of AS. We conducted this study. Methods to collect peripheral blood of active and stable AS patients, separate peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMCs), and to separate SYTOX Green-CD4+CD25-CD45RA+ T cells from PBMCs by flow cytometry. Cells) and SYTOX Green-CD4+CD25high Treg cells. The first part detected the proliferation, apoptosis and the ability to induce the differentiation of Tn cells to CD4+T cell subgroup Th17 cells. The selected Tn cells were labeled with 5 (6) - carboxyl fluorescein succinimide (5,6-carboxyfluorescein succinimidyl ester, CFSE) and added to the magnetic bead culture. Cultured cells were collected for fifth days in cell culture and Tn cells were detected and analyzed by flow cytometry. Tn cells were selected and cultured with anti -CD3/CD28 magnetic beads. Cells were collected for 24 hours and 72 hours in cell culture. Cells were stained with Annexin V and PI. The early and late apoptotic Tn were detected and analyzed by flow cytometry in 15 minutes. Cell ratio, PBMCs, flow cytometry was used to detect the expression of IL-23R, and the selected Tn cells were selected, and the recombinant human interleukin 6 (recombinant human interleukin 6, rhIL-6) was induced by the anti -CD3/CD28 magnetic beads and Th17 cells, and the recombinant human factor beta 1 (recombinant human transforming) was transformed. HTGF- beta 1), recombinant human interleukin 1 beta (recombinant human interleukin 1 beta, rhIL-1 beta), recombinant human interleukin 23 (recombinant human interleukin23, rhIL-23) culture, and cell culture third days and seventh days to detect the induction of differentiation ratio of Th17 cells. Second part used cell flow staining method to analyze Treg cells in PBMCs. The proportion of cell subgroups and their corresponding average fluorescence intensity (mean fluorescence intensity, MFI). Taking PBMCs, adding appropriate amount of corresponding cells, intracellular and nuclear flow antibody staining, Treg cells were detected and analyzed by flow cytometry, the proportion of Th17/Th1 cells and the corresponding MFI. third parts were used to detect the proliferation of Treg cells to Tn cells. Inhibition function. The selected Tn cells were selected and cultured separately or with the selected Treg cells, at the same time, the -CD3/CD28 magnetic beads were added to the cells. The cells were collected for fifth days in cell culture. The inhibitory function of Treg cells to the proliferation of Tn cells was detected by flow cytometry, and the enzyme linked immunoassay (enzyme-linked immunosorbent Assa) was used. Y, ELISA) method was used to detect the transformation of cell factor beta (transforming growth factor beta, TGF- beta), B (Granzyme B), interleukin 10 (interleukin 10), and interleukin 2 (2, 2) in co culture supernatant of Treg cells and Tn cells. Tn cells were selected separately or co cultured with Treg cells, and -CD3/CD28 beads were added to the cells. Cells were collected for fifth days to collect cells, and mRNA was extracted and reverse transcribed to cDNA. The real-time quantitative PCR (Real-Time quantitative polymerase chain reaction, qPCR) was used to detect the expression. And the expression of Treg cell related cytokine m RNA. Meanwhile, the selected Treg cells were selected to detect the phosphorylation level of the signal transduction and transcription activator 5 (signal transducer and activator of transcription 5, STAT5) at different time points after IL-2 stimulation. Rved non-coding DNA sequence, CNS) 2 epigenetic regulation is abnormal. Select the selected Treg cells, extract genomic DNA, and use Sodium Bisulfite sequencing to analyze the level of the 10 Cp G island methylation of the CNS2 region of the FoxP3 gene. Results 1. compared with healthy controls, the proliferation of Tn cells in active AS patients, apoptosis and the differentiation function to CD4+T cell subgroup Th17 cells were not significantly altered by.2. and healthy controls. There was no significant change in the proportion of Treg cells, Th17/Th1 cells and CD4 +T cell subgroups in PBMCs and Th17/Th1 cells in both stable AS patients and active AS patients. The Treg cell FoxP3 MFI decreased significantly in the patients with sex AS, suggesting that the Treg cell dysfunction.3. was not effective in inhibiting the proliferation of Tn cells in the active AS patients, indicating that the Treg cell function of the active AS patients was less than that of the healthy controls. At the same time, the qPCR test showed that the Tn cells in AS patients did not produce more IL-2 than the healthy control Tn cells, and the use of IL-2 in the co culture system did not decrease. These results suggest that the Treg cells in the active AS patients may have IL-2 utilization defects, which leads to the IL-2 decrease in the use of AS. Further detection of IL-2 stimulation. The level of phosphorylation of STAT5 in Treg cells at different time points showed that STAT5 expression in Treg cells increased after IL-2 stimulation in active AS patients, but the phosphorylation level did not increase correspondingly, which indicated that STAT5 phosphorylation in Treg cells was deficient or unstable. The above results showed that the IL-2 signaling pathway of Treg cells in active AS was indeed different. Often, this may affect the abnormal epigenetic regulation of the FoxP3 gene in the Treg cell of the Treg cell function.5. active AS patients, and the level of methylation in the Cp G island of the CNS2 region is elevated. Cell proliferation, apoptosis and the ability to induce differentiation to CD4+T cell subgroup Th17 cells were not found to be abnormal. We further analyzed the proportion of Treg cells and Th17/Th1 cells in the CD4+T cell subgroup of PBMCs in AS patients and the corresponding MFI, and first found that Treg fine cell FoxP3 MFI decreased significantly in the active AS patients, suggesting the function of the cells. Therefore, we further examined the proliferation inhibition of Treg cells to Tn cells, and found that Treg cells in active AS patients did not effectively inhibit the proliferation of Tn cells. These results showed that Treg cells in active AS patients did have functional defects. On the basis of the discovery of functional defects in Treg fine cells in active AS patients, this topic was introduced. One step is to focus on the possible mechanism of Treg cell dysfunction. On the one hand, we detected the IL-2 utilization of Treg cells in active AS and the abnormal downstream signal pathway of IL-2. It was found that Treg cells use the IL-2 ability to decrease, and the IL-2 in the environment is not well used when IL-2 is sufficient, and the important signal molecules downstream of IL-2 are STAT5. On the other hand, we detected the FoxP3 gene epigenetic regulation of Treg cells in active AS patients, and found that the abnormal elevated level of CpG island methylation in the CNS2 region and the increase of.IL-2 signaling pathway and the elevated level of CpG island methylation resulted in the abnormal inhibitory function of Treg cells, which eventually led to the occurrence of AS. At present, targeted Treg cells are in the ascendant for the treatment of autoimmune diseases and inflammatory diseases, and have made breakthrough progress. At present, different methods have been used to restore the number and function of Treg cells and to treat systemic lupus erythematosus (systemic lupus erythematosus, SLE), MS, psoriasis and ulcerative colitis (ulcerative colitis, UC). The findings of this study provide a theoretical basis for the clinical targeting of the recovery of Treg cell function for the treatment of AS, and also provide more directions and options for the treatment of AS from the underlying mechanism.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R593.23
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本文編號(hào)：1967975